Project Summary Cilia are cellular organelles that are essential for human development and health. It has long been known that cilia are organized into structurally and functionally distinct compartments known as the basal body, the transition zone, and the cilia shaft. Nephronophthisis-related ciliopathy (NPHP-RC) proteins localize to subregions within the previously known compartments, revealing a hidden complexity. For example, NPHP2/Inversin localizes to a proximal region of the ciliary shaft called the Inversin Compartment (InvC) that is not identifiable by any ultrastructural features. Despite the profound medical importance of cilia in human health and disease, how the ciliary shaft is spatially and functionally organized remains poorly understood. Identifying mechanisms controlling cilia shaft compartmentalization and understanding the physiological relevance of ciliary territories will be important in identifying therapeutic targets to combat cystic kidney diseases and other ciliopathies. The InvC is conserved in the nematode C. elegans, suggesting that the logic underlying the establishment of the InvC and ciliary compartmentalization is similar in worm and human cilia. In C. elegans, we found that the InvC regulates microtubule patterning and tubulin glutamylation. We also discovered that the Tubulin Code ? via tubulin isotypes and tubulin post-translational modifications ? sculpts ciliary structure, ciliary motor-based transport, and ciliary functions including release of ciliary extracellular vesicles. In this new application, we use C. elegans, an exceptional model for ciliary biology and human ciliopathies, to address the question of how the cilium is spatially and functional organized. First, we will define the origin and function of the InvC and examine the relationship between the InvC and tubulin glutamylation. Second, we determine how the Tubulin Code regulates microtubule ultrastructure, motor-based intraflagellar transport, and specialized ciliary functions. Finally, we will identify new genes and pathways that control ciliary homeostasis and protect against ciliary degeneration. This research will address the medically relevant question of how cilia are structurally and functionally organized in healthy and diseased states, and will provide fundamental insight to the molecules, mechanisms, and functions of ciliary compartmentalization and the Tubulin Code. These studies have direct implications for cystic kidney disease research because many of the genes and pathways explored in our work are associated with ciliopathies.